Juanchich et al. BMC Genomics (2018) 19:928 https://doi.org/10.1186/s12864-018-5344-z RESEARCHARTICLE Open Access Functional genomics of the digestive tract in broilers Amélie Juanchich1* , Christelle Hennequet-Antier1, Cédric Cabau2, Elisabeth Le Bihan-Duval1, Michel J. Duclos1, Sandrine Mignon-Grasteau1 and Agnès Narcy1 Abstract Background: The sustainability of poultry farming relies on the development of more efficient and autonomous production systems in terms of feed supply. This implies a better integration of adaptive traits in breeding programs, including digestive efficiency, in order to favor the use of a wider variety of feedstuffs. The aim of the project was to improve the understanding of genes involved in digestive functions by characterizing the transcriptome of different sections of the digestive tract: the junction between the proventriculus and the gizzard, the gizzard, the gastroduodenal junction, and the jejunum. Results: Total RNA from the four tissues were sequenced on a HiSeq2500 for six 23-day-old chickens from a second generation (F2) cross between two lines that were divergent for their digestive efficiency (D+/D-). Bioinformatics and biostatistics analyses of the RNA-seq data showed a total of 11,040 differentially expressed transcripts between the four tissues. In total, seven clusters of genes with markedly different expression profiles were identified. Functional analysis on gene groups was performed using “Gene Ontology” and semantic similarity. It showed a significant enrichment of body immune defenses in the jejunum, and an enrichment of transcriptional activity in the gizzard. Moreover, an interesting enrichment for neurohormonal control of muscle contraction was found for the two gizzard’s junctions. Conclusion: This analysis allows us to draw the first molecular portrait of the different sections of the digestive tract, which will serve as a basis for future studies on the genetic and physiological control of the response of the animal to feed variations. Keywords: Chicken, Broiler, Digestive tract, Transcriptome, Gizzard, Intestine Background understanding of adaptation processes - especially those Feed has represented the major proportion of production related to digestive efficiency - in order to improve poultry costs for meat-type chickens in recent years [1]. Addition- breeding schemes. Using high quality feedstuff, which are ally, the increasing demand for poultry meat and easily digested by all birds, does not make it possible to consequently for crops has accentuated the competition distinguish birds with a high or a low capacity for diges- between animal and human consumption. Poultry breed- tion. Feeding birds with wheat-based diets instead of ing has until now favored highly performing animals that corn-based diets is a way to challenge their digestive effi- also need high quality resources and an optimized produc- ciency in order to characterize their ability to digest vari- tion environment to express their genetic potential. ous types of feedstuffs. A divergent selection experiment Currently, the evolution towards more sustainable live- on the digestive efficiency of the chicken [2]usinga stock systems implies limiting inputs and to making use wheat-based challenge diet led to marked differences in of the adaptive capacity of the animals to changing and morphology and histology of the gizzard and small intes- even unfavorable dietary conditions. This requires a better tine [3]. The transit time between the different sections of * Correspondence: [email protected] 1BOA, INRA, Université de Tours, 37380 Nouzilly, France Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Juanchich et al. BMC Genomics (2018) 19:928 Page 2 of 9 the digestive tract may also explain differences in digestive TopHat2 [7] was used to align 89% of the reads to the efficiency: for instance particles, regardless of the size, Galgal4 version of the chicken genome. In total, 56,469 spent 10 times less time in the gizzard of birds with low transcripts were reconstructed using the Cufflinks tool digestive capacity compared to birds with high digestive [8] and then quantified using featureCounts [9]onall capacity [4]. The size and weight of the gizzard and the je- 24 samples. A total of 15,396 transcripts were consid- junum are highly different between birds as well [5, 6]. ered to be expressed in at least one of the four tissues. These results suggest that several functions are expected Biostatistical analyses with the edgeR package from the to be involved in the control of digestive efficiency. Bioconductor project [10, 11]revealedthatnotran- Theobjectiveoftheprojectwasthustoidentifygenesas- scripts were found to be differentially expressed be- sociated with underlying mechanisms by characterizing the tween animals with high or low digestive efficiency in transcriptome of key specialized sections of the digestive any of the four tissues. Therefore, a focus on the differ- tract: the gizzard (grinding and pre-digestion activity), the je- ences between tissues was made and biostatistical junum (major nutrient absorption site), and the junctions at analyses with the edgeR package revealed 11,040 differ- the entrance and exit of the gizzard (regulation of motility, entially expressed (DE) transcripts between the four tis- secretion and trophic activity of the digestive tract). The sues, by pairwise comparisons. The transcriptomic identification of genes and networks of genes involved in analysis confirmed that the four tissues (I: isthmus, G: digestive processes is a prerequisite for understanding this gizzard, GD: gastro-duodenal junction, and JE: je- complex biological function. These results will facilitate the junum) were clearly different compared to one another taking into account of digestive genetics in selection (Fig. 1a), and that the jejunum was the most different schemes through the future identification of genetic markers tissue compared to the three others. The homogeneity or biomarkers of feed efficiency. This will also be useful for within each tissue was high, especially for the gizzard the evaluation of new breeding or feeding systems. and the jejunum. The higher heterogeneity of the two junctions could partly result from the technical diffi- Results culty associated with their dissection. This descriptive Expression profiles of the digestive tract genes analysis also prompted us to exclude two samples from Transcriptome analysis by RNA-seq one individual, which were clearly mislabeled (the isth- Sequencing of the 24 samples on Hiseq2500 generated mus and gastro-duodenal junction were possibly between 5.2 and 10 million sequences per sample. switched). Pairwise comparisons showed a significant AB Fig. 1 Transcriptome analysis: overlap between samples. a Multi-Dimensional Scaling (MDS) plot showing similarity between samples. Numbers refer to individuals (144, 220, 235, 8071, 8196, and 8375) and letters to tissues (I: isthmus, G: gizzard, GD: gastro-duodenal junction, and JE: jejunum). b Venn diagram showing the number of transcripts differentially expressed that are common between two or more comparisons (I vs G: isthmus vs gizzard, G vs GD: gizzard vs gastro-duodenal junction, and GD vs JE: gastro-duodenal junction vs jejunum) Juanchich et al. BMC Genomics (2018) 19:928 Page 3 of 9 number of genes differentially expressed between the which represented 83% of the differentially expressed four tissues. A total of 1273 transcripts were common transcripts (9156/11,040). This strong impact of the je- between all pairwise comparisons. The Venn diagram junum in the results is consistent with the already in Fig. 1b focused on the spatio-temporal comparisons known differences in functions between the four stud- (I vs. G, G vs. GD, and GD vs. JE) and showed that ied tissues. Indeed, the main function of the gizzard is 1183 transcripts (i.e. 11% of the total) were differen- mechanical grinding and pre-digestion of the feed, tially expressed in the three comparisons. An increasing while the junctions upstream and downstream of the number of differentially expressed genes were found gizzard regulate the transit of feed into the gizzard. In while moving forward in the digestive tract: 2064 tran- contrast, the main function of the jejunum is the ab- scripts were differentially expressed between the isth- sorption of nutrients and immune system activity. Hier- mus and the gizzard, 6514 between the gizzard and the archical clustering also revealed specific signatures of gastro-duodenal junction and 9182 between the genes in the junctions upstream and downstream of the gastro-duodenal junction, and the jejunum. This could gizzard. Clusters 3 and 4 in particular contained 1064 be linked to the evolution of the biological function of genes specifically overexpressed in these two sections. these tissues, with the jejunum having a different func- The last two clusters grouped together